Plasma biomarker tool for the diagnosis of liver cancer comprising liver carboxylesterase 1 and liver cancer screening method

ABSTRACT

The present invention relates to a plasma biomarker for diagnosing hepatocellular carcinoma (HCC), in particular to the discovery of a protein in plasma using 2-D fluorescence differential gel electrophoresis (2-D DIGE), immunoprecipitation and Nano-liquid chromatography mass spectrometry (Nano-LC-MS/MS) system that was unknown on the basis of conventional techniques. By demonstrating the presence of liver carboxylesterase 1 (hCE1) in human plasma and confirming that its secretion level is higher in patients with HCC than in healthy volunteers, this invention may be used as a screening method to diagnose HCC at an early stage.

TECHNICAL FIELD

The present invention pertains to a plasma biomarker(carboxylesterase 1) for the diagnosis of liver cancer (hepatocellularcarcinoma; HCC), and the associated screening method for determiningdifferential biomarker expression between healthy volunteers andpatients with HCC employing 2-dimensional fluorescence gelelectrophoresis (2-D DIGE), immunoprecipitation and nano liquidchromatography mass spectrometry (Nano-LC-MS/MS) system. The amount ofbiomarker protein secreted into plasma is measured and used as anindicator of the presence of HCC.

BACKGROUND ART

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwideand has the fourth highest mortality rate. It is an especially majorproblem among Asian and African populations. Unlikely patients withother cancers, such as lung cancer and breast cancers, more than 95% ofHCC patients die within five years of being diagnosed with HCC.

Although HCC is the subject of continuing investigation and its symptomsare well known, early-stage diagnosis of this disease remains difficultand the survival rate after diagnosis is very low (3%-5%).

In addition to tissue biopsies, which are used for diagnosing HCC bycomputed tomography (CT) and magnetic resonance imaging (MRI), bodilyfluids, such as plasma, provide a clinical sample that allows for thesimultaneous measurement of proteins to determine the possible presenceof HCC.

To date, the biomarkers alpha-fetoprotein (AFP),des-gamma-carboxyprothrombin (DCP), glypican-3 (GPC3),alpha-1-fucosidase and transforming growth factor-b1 have been used,alone or in combination, for the clinical screening of HCC patients.Although these biomarkers are useful for the detection of HCC, theysuffer from poor sensitivity and/or specificity. For example,alpha-fetoprotein, which has been used as a serum marker for HCC formany years, has low sensitivity (39%-65%) and moderate specificity(76%-94%). Thus, there is an urgent need for a new class of biomarkerswith enhanced sensitivity and specificity, and which are capable ofdiagnosing HCC at an early stage.

The inventors of the present invention have discovered an N-linkedglycosylated protein that is a specific biomarker for HCC. This protein,human liver carboxylesterase 1 (hCE1), was identified by 2-dimensionalgel electrophoresis (2-D DIGE) and nano-liquid chromatography massspectrometry (Nano-LC-MS), and found to be differentially expressed inclinical plasma samples obtained from healthy volunteers and HCCpatients.

The candidate HCC biomarker protein, hCE1, which is expressed mainly inthe liver, is known to be expressed at lower levels in HCC tissuesrelative to normal liver tissue. However, there are no reports on thedirect detection of secreted hCE1 protein in human bodily fluid (i.e.,plasma). Consistent with this, searches of the public plasma proteindatabase (www.plasmaproteomedatabase.org) by the inventors have failedto uncover evidence for hCE1 in plasma.

Previous studies related to hCE1 in human plasma have used enzymaticassays employing the non-specific substrates, triolein or p-nitrophenylacetate, to measure the activity of hCE1 or its isoforms in samplespurified by sepharose-affinity chromatography. However, it has recentlybeen reported that this hydrolytic activity is likely attributable tothe esterase activity of butyrylcholinesterase, paraoxonase and albumin,which are also present in human plasma. This interpretation is supportedby the observation that the specific hCE1 inhibitor,bis-p-nitrophenylphosphate (BNPP), failed to confirm the presence ofhCE1 in human plasma.

In this context, the inventors have used proteomics techniques to detecthCE1 in both liver tissue and plasma. Using immunoprecipitation and theproteomics techniques, 2-D DIGE and Nano-LC-MS/MS system, the inventorshave demonstrated that hCE1 is present in the plasma of both healthyvolunteers and patients with HCC, and provided evidence that hCE1represents a novel HCC biomarker.

DISCLOSURE OF INVENTION Technical Problem

The object of the present invention is to provide new evidence that hCE1is a diagnostic biomarker for HCC that can effectively detect thepresence of HCC using patient plasma and a routine detection method.

The HCC screening method provided by the present invention comprises thesteps necessary to confirm the presence of hCE1 in a human blood sample,and discriminate between an patient with HCC and a healthy volunteerthrough quantitative measurement of hCE1 in plasma usingimmunoprecipitation, Western blot analysis and Nano-LC-MS/MS system.

Technical Solution

To achieve the above objective, the present invention verifies using 2-DDIGE and Western blot analysis that hCE1 expression is remarkablyreduced in tumorous liver tissue compared to non-tumorous liver tissueof a patient with HCC.

The present invention also uses proteomic techniques (Nano-LC-MS/MSsystem) to establish that hCE1 is, in fact, a plasma protein, somethingthat had not been previously shown using conventional methods.Furthermore, the present invention shows that the level of hCE1 proteinis 2-5 fold higher, on average, in the plasma of patients with HCC thanin that of healthy volunteers.

Because hCE1 is an N-linked glycoprotein, which binds lectin, thepresent invention employed lectin-affinity chromatography forglycoprotein separation in the analysis of non-tumorous and tumorousliver tissue. This was followed by 2-D DIGE, which localized hCE1protein to a position on the 2-D map that corresponded to a molecularweight of approximately 62 kDa and a PI of 4.5-5.3.

With the methods described in the present invention, the presence ofhCE1 was validated using immunoprecipitation and a highly-sensitiveNano-LC-MS/MS-based method after enrichment on magnetic beads(Dynabeads, Invitrogen). During the course of establishing optimalDynabeads-aliquot and plasma-concentration conditions, it was alsodetermined that a comparative analysis of the levels of hCE1 in plasmafrom healthy volunteers and patients with HCC could be accomplishedusing Western blot analyses.

According to the present invention, the presence of hCE1 in human plasmacan be confirmed through the use of proteomic technology by applying thefollowing steps:

a) Collect a blood sample into a tube containing di-potassiumethylenediamine tetraacetic acid (K₂EDTA) and maintain for 30 minutes atroam temperature; centrifuge for 15 minutes at 2,400×g to obtain plasma(supernatant);

b) Combine 400 μg of anti-hCE1 antibody with 10 mg of magnetic beads,then transfer 500 μg of the antibody-coated magnetic beads to a 1 mltube containing 8 mg plasma from HCC patients or healthy volunteers andincubate for 2 hours to immunoprecipitate hCE1 and separate it from theremaining plasma proteins;

c) Denature each sample of immunoprecipitated hCE1 from healthyvolunteers and HCC patients in lysis buffer, and separate the samplesusing 1-dimensional gel electrophoresis;

d) Excise the gel band at approximately 62 kDa, and digest the proteininto peptides using trypsin; and

e) Analyze the peptide digest using high-sensitivity Nano-LC-MS/MSsystem to identify hCE1 protein (See FIG. 6 and FIG. 7).

Furthermore, the level of secreted hCE1 in human plasma is also measuredin the present invention and can be used as a biomarker fordiscriminating between an patient with HCC and a healthy volunteer usingthe following series of steps:

1) Confirm that the level of hCE1 expression in tumorous liver tissuefrom a patient with HCC is markedly decreased compared to non-tumorousliver tissue using 2-D DIGE and Western blot analysis;

2) Immunoprecipitate a plasma sample as described above steps a-b) toobtain a solution of hCE1;

3) Confirm the presence of hCE1 signal by Western blotting analysisusing an anti-hCE1 antibody; and

4) Verify that the hCE1 signal is stronger in the plasma of patientswith HCC than in that of the healthy volunteers (See FIG. 8 and FIG. 9).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph showing a 2-D DIGE image of fractionatedglycoproteins from non-tumorous liver tissue from a patient with HCC;the position of hCE1 is indicated.

FIG. 2 is a photograph showing a 2-D DIGE image of fractionatedglycoproteins from tumorous liver tissue from a patient with HCC; theposition of hCE1 is indicated.

FIG. 3 is a photograph showing the differences in hCE1 expression levelsin paired samples of non-tumorous and tumorous liver tissue fromadditional ten patients with HCC.

N: Non-tumorous liver tissue

T: Tumorous liver tissue

FIG. 4 is a photograph of an immunohistochemically stained,paraffin-embedded tissue array constructed from paired non-tumorous andtumorous liver sections from 47 patients with HCC showing the level ofhCE1 expression.

FIG. 5 is a graph depicting the frequency distribution of stainingscores determined by immunohistochemical staining in paired non-tumorousand tumorous liver sections of patients with HCC. The results indicatethat the level of hCE1 expression is specifically reduced in thetumorous liver sections compared to the non-tumorous liver sections.

FIG. 6 is a table showing the tryptic peptides of hCE1 identified byNano-LC-MS/MS system in the plasma of healthy volunteers and patientswith HCC, and used to validate the presence of hCE1 in human plasma.

FIG. 7 is a representative Nano-LC-MS/MS chromatograph of the commonpeptides shown in FIG. 6.

FIG. 8 is a photograph showing the differences in hCE1 levels betweenrepresentative three healthy volunteers and three patients with HCC,determined using Western blot analysis after immunoprecipitation.

Np: plasma of healthy volunteer

Tp: plasma of patient with HCC

FIG. 9 is a graph comparing the hCE1 levels in the plasma of eighthealthy volunteers and eight patients with HCC.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is described in detail in the followingembodiments. The embodiments are only included to describe the presentinvention, however, the scope of the present invention is not limited tothe embodiments.

Embodiment 1: Collection of Clinical Tissues and Plasma

The non-tumorous and tumorous liver tissue from patients with HCC wereobtained along with pathological individual information from theDepartment of Pathology, Yonsei University College of Medicine, Seoul,Korea. The following tests were used to assess the appropriateness ofplasma from healthy volunteers to serve as a negative control: HIV-1 andHIV-2 antibodies derived from HIV, which is a liver-cancer indicatingstandard test element; HIV-1 antigen; hepatitis B surface antigen;hepatitis B core antigen; hepatitis C virus; T-Cell Leukemia Virus(HTLV-I/II) antigen; and Treponema pallidum. Plasma was obtainedaccording to a standardized sample separation method formally adopted bythe Human Proteome Organization (HUPO).

Each blood sample (4 ml) from man or woman was maintained in a tubecontaining di-potassium ethylenediamine tetraacetic acid (K₂EDTA) for 30minutes at room temperature, followed by centrifugation for 15 minutesat 2,400×g. A supernatant (plasma) was retained. Samples ofnon-tumorous, tumorous liver tissue, and plasma were stored at −70° C.until ready for use. Liver tissues and plasma were acquired according toapproved procedures of the Institutional Review Board (IRB) at YonseiUniversity College of Medicine with informed consent of the patients.

Embodiment 2: Separation of Glycoproteins in Non-Tumorous and TumorousLiver Tissue

A 100 mg sample of each tissue was homogenized in RIPA buffer (50 mMTris, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, pH 7.4)at 4° C. Glycoproteins were separated by lectin-affinity chromatographyusing a mixture of five different agarose-bound lectins: concanavalin A,wheat germ agglutinin, Jacalin, Sambucus nigra, and Aleuria aurantia.The lectin mixture, with binding specificities for glycoproteins withdifferent sugar compositions, was packed into a 2 ml PD-10 column(Pierce). Glycoprotein-containing samples were applied to the column inbinding buffer (20 mM Tris, 1 mM MnCl₂, 1 mM CaCl₂, 0.15 M NaCl, pH 7.4)and allowed to interact with the column for 30 minutes.

The bound glycoproteins were eluting with a buffer (0.2 Mmethyl-D-mannopyroside, 0.2 M methyl-D-glucopyroside, 0.2 MN-acetylglucosamine, 0.2 M galactose, 0.1 M lactose, 0.1 M fucose, 20 mMTris, 0.5 M NaCl, pH 7.0) that displaces glycoproteins on the basis ofsugar composition. The eluted glycoproteins were concentrated using a5-kDa membrane filter, and precipitated with 50% trichloroacetic acidand 100% ice-cold acetone. The precipitate was dissolved in 2-D lysisbuffer (7 M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris, pH 8.5), and theresulting protein solution was adjusted to pH 8.5. The concentration ofthe protein was measured using a 2D Quant kit (GE Healthcare).

Embodiment 3: 2-Dimensional Fluorescence Differential GelElectrophoresis (2-D DIGE)

Ten paired samples (five from non-tumorous liver tissue and five fromtumorous liver tissue) from five patients were used to prepare analyticsamples (50 μg each) for 2-D DIGE. Analytic samples and a correspondingpooled standard sample were labeled individually with the fluorescentdyes, Cy3, Cy5 and Cy2 (400 pmol; GE Healthcare) in the dark for 30minutes.

The reaction was stopped by adding 1 μl 10 mM lysine, and the threelabeled samples were mixed, adjusted to a final volume of 450 μl byadding sample buffer (6 M urea, 2 M thiourea, 4% CHAPS, 60 mMdithiothreitol (DTT), 30 mM Tris, pH 8.5) and rehydrated by incubatingwith a 2% IPG 3-10 NL buffer solution for 16 hours at room temperature.

Isoelectric focusing (IEF) was performed using the Immobiline DryStrippH 3-10 NL under optimized conditions (up to 95,000 V) on the MultiPhorII electrophoresis system (GE Healthcare), followed by a one-stepreduction and alkylation in which the DryStrip was incubated for 25minutes in a tributylphosphine (TBP) buffer solution (6 M urea, 2%sodium dodecyl sulfate [SDS], 30 mM Tris, 20% glycerol, 2.5% acrylamidesolution, 5 mM TBP).

After reduction and alkylation, the isoelectrically focused proteinswere separated in the second dimension by electrophoresis on 9%-16%gradient polyacrylamide gels using an Ettan Dalt-twelve electrophoresissystem (GE Healthcare). Each gel was then scanned at wavelengthscorresponding to Cy2, Cy3 and Cy5 using a Typhoon 9400 (GE Healthcare)scanner, and images of each gel were analyzed using Decyder 2-D analysissoftware (GE Healthcare).

Embodiment 4: Identification of 2-D DIGE-Separated Proteins

After images were analyzed, a spot corresponding to a significantlydifferentially expressed protein was excised from a Coomassieblue-stained gel, destained and digested with trypsin. The digestedpeptides were desalted using a Poros R2 and Oligo R3 resin mixture. Theprotein was analyzed using a 4800 MALDI-TOF-MS (Applied Biosystem) andthe spectra were identified using a MASCOT database.

Embodiment 5: Validation of HCC Biomarker Expression Levels in Tissuesby Western Blot Analysis

Differences in HCC biomarker protein concentration between non-tumorousand tumorous liver tissue from ten patients were verified by Westernblotting analysis. An equal amount of protein (5 μg) from each samplewas separated by SDS-PAGE on 10% gels.

Proteins were then transferred to a nitrocellulose (NC) membrane, andblocked by incubating in TBS-T buffer (20 mM Tris, 137 mM NaCl, 0.1%Tween-20, pH 7.6) containing 5% skim milk for 1 hour at roamtemperature. Membranes were then incubated with primary anti-hCE1antibody (CE1, Abcam; 1:10000 in TBS-T/5% skim milk) for 1 hour at roomtemperature. A mouse β-actin antibody (Santa Cruz) was used as apositive control.

After washing with TBS-T/5% skim milk, membranes were incubated withsecondary horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (SantaCruz; 1:20000 dilution) for 1 hour at room temperature. Immunoreactiveproteins were detected using ECL Plus Western blot reagents (GEHealthcare), and blots were scanned and analyzed using a Typhoon 9400scanner.

Embodiment 6: Validation of HCC Biomarker Expression Levels in TissuesUsing an Immunohistochemical Staining Method

Paraffin-embedded tissue arrays constructed from paired tissues fromnon-tumorous and tumorous liver sections of 47 patients with HCC wereused for immunohistochemistry. The slides were deparaffinized withxylene and alcohol, hydrated, and then treated with 0.6% hydrogenperoxide. Anti-hCE1 antibody (1:100) was applied to the slide andincubated for 30 minutes. hCE1-bound antibody color was developed usingChemMate Envision Kit (DAKO) and colorized with hematoxylin (used as acontrast staining agent) for 30 seconds. The intensity of staining wasscored as 1) “−”, for no staining; 2) “+”, for weak staining; and 3)“++”, for strong staining.

Embodiment 7: Validation of HCC Biomarker Protein Secretion Level inHuman Plasma by Immunoprecipitation and Western Blot Analysis

Dynabead MyOne™ Tosylactivated (Invitrogen) magnetic beads were coatedwith anti-hCE1 antibody as described by the manufacturer. Briefly,magnetic beads (10 mg) and anti-hCE1 antibody (400 μg) were mixed andincubated for 16 hours in binding buffer (0.1 M sodium borate, 1 Mammonium sulfate, pH 9.5) at 37° C., and then blocked with TBS-T/5% skimmilk for an additional 16 hours. hCE1 was immunoprecipitated from theplasma of healthy volunteers and patients with HCC by transferringantibody-coated beads (500 μg) to 1 ml tubes containing 8 mg plasma andincubating for 2 hours.

Antibody-bound proteins were eluted by adjusting the pH of the TBS-Tbuffer to pH 2. Differences in hCE1 levels between the plasmas ofhealthy volunteers and patients with HCC were determined by Western blotanalysis using a primary anti-hCE1 antibody (Abcam; 1:1000) and asecondary anti-rabbit IgG-HRP antibody (Santa Cruz; 1:5000), asdescribed in Embodiment 5.

Embodiment 8: Validation of hCE1 Protein in Human plasma byNano-LC-MS/MS System

A band corresponding to hCE1 based on Western blot analysis (embodiment7) was excised from the gel, and reduced and alkylated bydithiothrietol(DTT)/iodoacetic acid (IAA) treatment. After digestingwith trypsin to yield peptides, the identity of the gel-isolated proteinas hCE1 was validated using Nano-LC-MS/MS system and a linear trapquadrupole (LTQ) detector.

INDUSTRIAL APPLICABILITY

As described above, hCE1 was discovered as a new biomarker for HCCdiagnosis. According to the present invention, hCE1 protein secretionlevel in plasma can function as an index that allows early diagnosis ofHCC, and thereby contributes to increasing the survival rate amongpatients with HCC by enabling timely therapeutic intervention.

The invention claimed is:
 1. A screening method for hepatocellularcarcinoma (HCC), comprising: collecting human blood, and detecting thepresence of human liver carboxylesterase 1(hCE1) protein in plasma ofthe human blood as a plasma biomarker for HCC diagnosis; wherein thelevel of hCE1 protein is increased more in the plasma of patients withHCC than in the plasma of healthy patients.
 2. The method of claim 1,wherein the level of hCE1 protein is increased, on average, 2-5 foldmore in the plasma of patients with HCC compared to the plasma ofhealthy patients.
 3. The method of claim 1, wherein the presence of thehCE1 protein is detected by an anti-hCE1 antibody.
 4. A method fordiagnosing hepatocellular carcinoma (HCC) in a subject, the methodcomprising the steps of: a) collecting a sample of human blood from thesubject; b) contacting a portion of the blood sample with an antibodyhaving specific binding affinity for human liver carboxylesterase 1(hCE1), thereby forming a complex between the antibody and hCE1, theantibody having a detectable label; c) separating the complex formed insaid contacting step (b) from labeled antibody not comprising thecomplex; d) quantifying a signal from the detectable label of theantibody comprising the complex formed in said contacting step (b), thesignal being proportional to an amount of hCE1 in the blood sample,whereby the amount of hCE1 in the sample is calculated; e) comparing theamount of hCE1 calculated in said quantifying step (d) to a hCE1reference amount; and f) providing a diagnosis of HCC in the subject ifthe amount of hCE1 in the sample calculated in said quantifying step (d)is greater than the hCE1 reference amount; wherein the hCE1 referenceamount is an amount of hCE1 in a blood sample from a subject not havingHCC.
 5. The method of claim 4, wherein the blood sample comprisesplasma.
 6. The method of claim 4, wherein the blood sample comprisesserum.
 7. The method of claim 4, further comprising the step ofseparating human liver carboxylesterase 1 (hCE1) protein from otherremaining proteins in the blood sample, wherein said separating hCE1protein step occurs between steps (a) and (b).
 8. The method of claim 7,wherein the hCE1 protein is separated from other remaining proteins inthe blood sample by immunoprecipitation.
 9. The method of claim 7,wherein the step of separating hCE1 protein from other remainingproteins in the blood sample comprises the following steps: i)contacting a portion of the sample from the subject with an antibodyhaving specific binding affinity for hCE1, thereby forming a complexbetween the antibody and hCE1; and ii) precipitating the complex formedin said contacting step (i); iii) separating the precipitated complexfrom the supernatant of the sample, the supernatant comprising the otherremaining proteins and antibody not comprising the complex; wherein thehCE1 protein is separated from the other remaining proteins in the bloodsample.
 10. The method of claim 9, wherein the antibody is linked to amagnetic molecule.
 11. The method of claim 4, wherein the hCE1 referenceamount is an amount of hCE1 in the blood of a healthy subject.